مقايسه روش هاي رديابي Armillaria mellea عامل پوسيدگي آرميلاريايي ريشه و طوقه درختان در خاك و گياه با استفاده از روش هاي سنتي و آغاز گرهاي اختصاصي

چكيده انگليسي :

Abstract Armillaria root and crown rot is a serious and important disease of woody plants The disease has a universaldistribution and causes economical losses on a broad host range of trees in gardens forests and urbanenvironments The aerial symptoms of disease are discoloration yellowing drying and defoliation of leaves anddie back of trees The signs of Armillaria root and crown rot include formation of white fan mycelium under thebark black rhizomorphs and occurrence of basidiocarps around the crown of infected trees The causal agent ofthe disease is a Basidiomycota fungus belongs to Tricholomataceae family and the genus Armillaria in whichthe species Armillaria mellea is well known among different species for its pathogenicity on trees Consideringthe importance of the disease detection of the pathogen from soils of gardens nurseries and urbanenvironments is necessary to establish on IPM program for decreasing and prevention of losses Numerousmethods have been used for the detection of soil born fungi including semi selective media baiting andserological and molecular methods To achieve a specific sensitive and rapid detection method for the pathogenin soil and root the present investigation was conducted Thirteen isolates belonging to genus Armillaria wereobtained via culturing basidiocarps growing around the crown of infected trees and confirmed using universalprimers ITS1 ITS4 and specific primers AR1 AR2 in nested PCR Species specific primers ITS4 AMEL3 wereused to identify A mellea and nine isolates were shown specific band of A mellea To study genetic diversity PCR RFLP of ITS region amplified with primers ITS4 AMEL3 was carried out using HinfI AluI and MseIrestriction enzymes Results showed that only HinfI could distinguish two groups within isolates of A mellea At the next step the isolates # 295c and A2 were used as inoculum that were previously identified as A melleaand was inoculated into the soil For detection of pathogen soil direct culture method baiting and nested PCRmethod were used simultaneously The culture medium was not capable of detecting pathogen in long termperiod In the baiting method Pelargonium hortorum rooted cutting was used as bait In this method requiredtime to detect pathogen was quite long In nested PCR method detection of pathogen was done successfully forisolates # 295c within six months and isolate #A2 within three months at different intervals To verify theaccuracy of the used detection method the nested PCR product was sequenced directly Results revealed thatthere is 99 similarity between Isfahan isolate and the isolate A mellea registered in GenBank Then thepossibility of pathogen detection in wood and soil samples which were collected from gardens and nurseries wastested with soil direct culture method and nested PCR in which only nested PCR method was successful andefficient To test the sensitivity of nested PCR reaction serial dilution of DNA from a soil sample was examinedand enabled the amplification of about one pg L The results revealed that among three different testeddetection methods nested PCR is an efficient method for the detection of A mellea in soil and root samples dueto its rapid detection sensitivity and specificity Key words Detection methods Root and crown rot Armillaria mellea Nested PCR

يوسفي همداني، الهام

مقايسه روش هاي رديابي Armillaria mellea عامل پوسيدگي آرميلاريايي ريشه و طوقه درختان در خاك و گياه با استفاده از روش هاي سنتي و آغاز گرهاي اختصاصي

Nested PCR